Impact of heme and heme degradation products on acute kidney injury associated with Shiga toxin 2-induced hemolytic-uremic syndrome

溶血性尿毒症综合征(HUS)是由肠出血性大肠杆菌(EHEC)感染引起的一种严重的肠外并发症。细菌性志贺毒素(STX)，尤其是STX2，通过引发血栓性微血管病变，继而引起溶血性贫血、血小板减少和急性肾损伤的典型三联体症状，在HUS的发病机制中发挥重要作用。最近，细胞外“游离”的血红素作为威胁生命的感染的永久因素的核心作用已经得到承认，即使是非常温和的溶血。然而，作为溶血性贫血伴发炎症的典范，溶血、“游离”的血红素和血红素降解产物(HHDP)以及血红蛋白和血红素中和蛋白和血红素分解代谢酶(血红素加氧酶1，HO1)对HUS急性肾损伤程度的影响有待研究。与其他形式的溶血性贫血形成鲜明对比的是，HUS患者的血清促红细胞生成素(EPO)水平也出现了平行的下降。这可能是由于抑制EPO基因表达而引起的炎症，或者是HUS特征的直接损害肾脏并伴有肾性贫血。众所周知，多效激素EPO具有器官保护作用，这与其公认的造血作用无关。越来越多的证据表明，EPO的组织保护作用是由促红细胞生成素受体和b共同受体(Bcr)的异源复合体介导的，这种bcr在药理上不同于表达红细胞生成的促红细胞生成素受体同源二聚体。因此，溶血/血红素依赖性损伤和EPO/bcr依赖性保护之间的严重失衡可能会加重HUS的肾功能障碍。了解这种不平衡具有开发新的治疗策略的潜力。在建议的子项目中，我们的目标是确定溶血/细胞外血红素和EPO/BCR信号轴在HUS相关性急性肾损伤发生中的作用。在翻译方法中，我们将专门研究(I)HHDP的溶血和形成的作用(Ii)血红蛋白和血红素清除蛋白的影响(Iii)HO1的影响，以及(Iv)EPO的影响和潜在的BCR介导的EPO对HUS肾功能障碍和损伤的组织保护作用。我们将在Stx2介导的实验性HUS模型中，通过使用缺乏结合珠蛋白、血凝素、HO1或BCR的小鼠来系统地解决这些目标。通过量化HUS患者血清样本中的HHDPs和EPO，并将结果与这些患者的肾功能障碍的程度/结果相关联，这些机制的小鼠研究将得到补充。拟议的研究将使我们能够表征溶血/血红素和EPO/BCR信号轴在Stx2传播性肾功能障碍和损伤发病机制中的影响，Stx2传播性肾功能障碍和损伤是终末期肾脏疾病的重要危险因素。

The hemolytic-uremic syndrome (HUS) is a serious extra-intestinal complication of infections with enterohemorrhagic Escherichia coli (EHEC). Bacterial Shiga toxins (Stx), in particular Stx2, play an essential role in HUS pathogenesis by triggering thrombotic microangiopathy and subsequently the typical triad of symptoms of hemolytic anemia, thrombocytopenia and acute kidney injury. Recently, the central role of extracellular "free" heme as a perpetuating factor in lifethreatening infections, even with a very moderate degree of hemolysis, has been acknowledged. However, the impact of hemolysis, "free" heme and heme degradation products (HHDPs) as well as hemoglobin- and heme-neutralizing proteins and heme-catabolizing enzymes (heme oxygenase 1, HO1) on the degree of acute kidney injury in HUS, as a paradigm for hemolytic anemia with concomitant inflammation, requires investigation. In striking contrast to other forms of hemolytic anemia, a parallel decrease in erythropoietin (EPO) serum levels has been described in HUS patients. This might be due to either inflammation via inhibition of EPO gene expression or a HUS characteristic direct damage to the kidneys with concomitant renal anemia. The pleiotropic hormone EPO is known to have organ protective properties, which are independent of its well-established hematopoietic effects. There is increasing evidence that the tissue-protective effects of EPO are mediated by a heterocomplex of the erythropoietin receptor and the b-common receptor (bcR) that is pharmacologically distinct from the erythropoietin receptor homodimer conveying erythropoiesis. Thus, a critical imbalance between hemolysis/heme-dependent injury and EPO/bcR-dependent protection might aggravate renal dysfunction in HUS. Understanding this imbalance carries the potential for novel therapeutic strategies. In the proposed subproject we aim to establish the role of hemolysis/extracellular heme and the EPO/bcR signaling axis in the development of HUS-associated acute kidney injury. In a translational approach we will specifically investigate (i) the role of hemolysis and formation of HHDPs (ii) the impact of hemoglobin- and heme-scavenging proteins (iii) the impact of HO1, and (iv) the impact of EPO and the potential bcR-mediated tissue protective effects of EPO on renal dysfunction and injury in HUS. We will address these objectives systematically in a model of Stx2-mediated experimental HUS by employing mice deficient for haptoglobin, hemopexin, HO1 or bcR. These mechanistic murine studies will be complemented by quantification of HHDPs and EPO in serum samples from HUS patients and by correlating the results to the degree/outcome of renal dysfunction in these patients. The proposed studies will enable us to characterize the impact of hemolysis/heme and the EPO/bcR signaling axis in the pathogenesis of Stx2 propagated renal dysfunction and injury, a significant risk factor for end-stage kidney disease.